Parallel preparation of high fidelity probes in an array format

ABSTRACT

The present invention provides massively parallel oligonucleotide synthesis and purification for applications that utilize large collections of defined high-fidelity oligonucleotides (e.g., from about 10 1  to about 10 5  different sequences, generally between 25-160 bases in length).

FIELD OF THE INVENTION

The present invention relates generally to the fabrication ofoligonucleotides such as probes and primers using oligonucleotide arraytechnology. The present invention relates to massively paralleloligonucleotide synthesis and purification for applications that utilizelarge collections of defined high-fidelity oligonucleotides.

BACKGROUND OF THE INVENTION

PCR techniques are well-established and widely used across varioussegments of life-science research, diagnostics, etc. An increasinglyimportant trend in the application of PCR is the ability to multiplexthe reaction, which requires, in addition to the usual thermal cyclingequipment and enzyme, sets of carefully designed oligonucleotide primers(or probes). Oligonucleotide primers are traditionally prepared by thesolid-supported phosporanidite approach, either on controlled-poreglass, polymeric support or membrane support.

Following oligonucleotide assembly, the support is typically treatedwith a deprotection reagent to remove protecting groups and to cleavethe oligonucleotide from the support in a single step. Due to the highstepwise efficiency of the solid-supported phosphoramidite approach, itis often not necessary to rigorously purify short oligonucleotides(25-40 mers) destined for use as PCR primers. More often, simple ethanolprecipitation or cartridge separation is used to “desalt” the primer andremove small molecular-weight components.

Although careful purification is atypical, some means of identity andpurity confirmation (i.e., QC) are normally required and the collectionof such data is considered good lab practice. Primer confirmation canusually be accomplished by high-throughput analytical techniques such asMALDI-TOF mass spectrometry and/or capillary gel electrophoresis.Conventional small-scale solid-supported oligonucleotide synthesismethods (flow-through column, membrane, 96-well plate) produce enoughprimer for thousands of PCR reactions.

SUMMARY OF THE INVENTION

Methods are provided for generating high numbers of oligonucleotides asprobes and primers using oligonucleotide array technology to provideoligonucleotides, probes and primers.

In particular, methods are also provided for fabricating a plurality ofoligonucleotides having free 3′-hydroxyl groups from a high densityoligonucleotide array. In a preferred embodiment, synthesis is initiatedwith a reverse-orientation RNA monomer that contains an orthogonal 2′-OHprotecting group. Following conventional 3′→5′ probe synthesis, the2′-OH protecting group is removed to allow base-induced intramoleculartransesterification. The transesterification reaction causes release ofthe synthesized probe with an authentic 3′-hydroxy functionality, whilethe 2′,3′-cyclic phosphate remains attached to the solid support.

Another disclosed method has the steps of providing a solid substrate;attaching a plurality of linkers to the substrate, each said linkerhaving a cleavable moiety, wherein the cleavable moiety is activatableat a distinct set of conditions and wherein activation of the cleavablemoiety disrupts the linker to allow release of anything joined to thelinker at the site of the cleavable moiety, to provide a plurality ofattached linkers; attaching a first monomer to at least one of saidplurality of linkers to provide an attached first monomer; attaching asecond monomer to a least one of said attached first monomers or saidattached plurality of linkers to provide an attached second monomer;attaching a third monomers to a least one of said attached firstmonomer, second monomers or plurality of linkers to provide an attachedthird monomer; repeating said step of attaching a monomer until thedesired array of polymers is complete and subjecting the array to thedistinct set of conditions to provide release of polymers from thearray.

DESCRIPTION OF THE DRAWINGS

“FIG. 1” depicts the transesterification reaction causing release of thesynthesized probe with an authentic 3′-hydroxy functionality, while the2′,3′-cyclic phosphate remains attached to the solid support.

“FIG. 2” shows two exemplary monomers used in the process depicted inFIG. 1.

“FIG. 3” depicts a purification scheme to obtain full length probes withauthentic termi.

DETAILED DESCRIPTION OF THE INVENTION

A. General

The present invention has many preferred embodiments and relies on manypatents, applications and other references for details known to those ofthe art. Therefore, when a patent, application, or other reference iscited or repeated below, it should be understood that it is incorporatedby reference in its entirety for all purposes as well as for theproposition that is recited.

As used in this application, the singular form “a,” “an,” and “the”include plural references unless the context clearly dictates otherwise.For example, the term “an agent” includes a plurality of agents,including mixtures thereof.

An individual is not limited to a human being but may also be otherorganisms including but not limited to mammals, plants, bacteria, orcells derived from any of the above.

Throughout this disclosure, various aspects of this invention can bepresented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 3, 4, 5, and 6. This appliesregardless of the breadth of the range.

The practice of the present invention may employ, unless otherwiseindicated, conventional techniques and descriptions of organicchemistry, polymer technology, molecular biology (including recombinanttechniques), cell biology, biochemistry, and immunology, which arewithin the skill of the art. Such conventional techniques includepolymer array synthesis, hybridization, ligation, and detection ofhybridization using a label. Specific illustrations of suitabletechniques can be had by reference to the example herein below. However,other equivalent conventional procedures can, of course, also be used.Such conventional techniques and descriptions can be found in standardlaboratory manuals such as Genome Analysis: A Laboratory Manual Series(Vols. I-IV), Using Antibodies: A Laboratory Manual, Cells: A LaboratoryManual, PCR Primer: A Laboratory Manual, and Molecular Cloning: ALaboratory Manual (all from Cold Spring Harbor Laboratory Press),Stryer, L. (1995) Biochemistry (4th Ed.) Freeman, New York, Gait,“Oligonucleotide Synthesis: A Practical Approach” 1984, IRL Press,London, Nelson and Cox (2000), Lehninger, Principles of Biochemistry3^(rd) Ed., W.H. Freeman Pub., New York, N.Y. and Berg et al. (2002)Biochemistry, 5^(th) Ed., W.H. Freeman Pub., New York, N.Y., all ofwhich are herein incorporated in their entirety by reference for allpurposes.

The present invention can employ solid substrates, including arrays insome preferred embodiments. Methods and techniques applicable to polymer(including protein) array synthesis have been described in U.S. Ser. No.09/536,841, WO 00/58516, U.S. Pat. Nos. 5,143,854, 5,242,974, 5,252,743,5,324,633, 5,384,261, 5,405,783, 5,424,186, 5,451,683, 5,482,867,5,491,074, 5,527,681, 5,550,215, 5,571,639, 5,578,832, 5,593,839,5,599,695, 5,624,711, 5,631,734, 5,795,716, 5,831,070, 5,837,832,5,856,101, 5,858,659, 5,936,324, 5,968,740, 5,974,164, 5,981,185,5,981,956, 6,025,601, 6,033,860, 6,040,193, 6,090,555, 6,136,269,6,269,846 and 6,428,752, in PCT Applications Nos. PCT/US99/00730(International Publication No. WO 99/36760) and PCT/US01/04285(International Publication No. WO 01/58593), which are all incorporatedherein by reference in their entirety for all purposes.

Patents that describe synthesis techniques in specific embodimentsinclude U.S. Pat. Nos. 5,412,087, 6,147,205, 6,262,216, 6,310,189,5,889,165, and 5,959,098. Nucleic acid arrays are described in many ofthe above patents, but the same techniques are applied to polypeptidearrays.

Nucleic acid arrays that are useful in the present invention includethose that are commercially available from Affymetrix (Santa Clara,Calif.) under the brand name GeneChip®. Example arrays are shown on thewebsite at affymetrix.com.

The present invention also contemplates many uses for polymers attachedto solid substrates. These uses include gene expression monitoring,profiling, library screening, genotyping and diagnostics. Geneexpression monitoring and profiling methods can be shown in U.S. Pat.Nos. 5,800,992, 6,013,449, 6,020,135, 6,033,860, 6,040,138, 6,177,248and 6,309,822. Genotyping and uses therefore are shown in U.S. Ser. Nos.10/442,021, 10/013,598 (U.S. Patent Application Publication20030036069), and U.S. Pat. Nos. 5,856,092, 6,300,063, 5,858,659,6,284,460, 6,361,947, 6,368,799 and 6,333,179. Other uses are embodiedin U.S. Pat. Nos. 5,871,928, 5,902,723, 6,045,996, 5,541,061, and6,197,506.

The present invention also contemplates sample preparation methods incertain preferred embodiments. Prior to or concurrent with genotyping,the genomic sample may be amplified by a variety of mechanisms, some ofwhich may employ PCR. See, for example, PCR Technology: Principles andApplications for DNA Amplification (Ed. H. A. Erlich, Freeman Press, NY,N.Y., 1992); PCR Protocols: A Guide to Methods and Applications (Eds.Innis, et al., Academic Press, San Diego, Calif., 1990); Mattila et al.,Nucleic Acids Res. 19, 4967 (1991); Eckert et al., PCR Methods andApplications 1, 17 (1991); PCR (Eds. McPherson et al., IRL Press,Oxford); and U.S. Pat. Nos. 4,683,202, 4,683,195, 4,800,1594,965,188,and 5,333,675, and each of which is incorporated herein byreference in their entireties for all purposes. The sample may beamplified on the array. See, for example, U.S. Pat. No. 6,300,070 andU.S. Ser. No. 09/513,300, which are incorporated herein by reference.

Other suitable amplification methods include the ligase chain reaction(LCR) (for example, Wu and Wallace, Genomics 4, 560 (1989), Landegren etal., Science 241, 1077 (1988) and Barringer et al. Gene 89:117 (1990)),transcription amplification (Kwoh et al., Proc. Natl. Acad. Sci. USA 86,1173 (1989) and WO88/10315), self-sustained sequence replication(Guatelli et al., Proc. Nat. Acad. Sci. USA, 87, 1874 (1990) andWO90/06995), selective amplification of target polynucleotide sequences(U.S. Pat. No. 6,410,276), consensus sequence primed polymerase chainreaction (CP-PCR) (U.S. Pat. No. 4,437,975), arbitrarily primedpolymerase chain reaction (AP-PCR) (U.S. Pat. Nos. 5,413,909, 5,861,245)and nucleic acid based sequence amplification (NABSA). (See, U.S. Pat.Nos. 5,409,818, 5,554,517, and 6,063,603, each of which is incorporatedherein by reference). Other amplification methods that may be used aredescribed in, U.S. Pat. Nos. 5,242,794, 5,494,810, 4,988,617 and in U.S.Ser. No. 09/854,317, each of which is incorporated herein by reference.

Additional methods of sample preparation and techniques for reducing thecomplexity of a nucleic sample are described in Dong et al., GenomeResearch 11, 1418 (2001), in U.S. Pat. No. 6,361,947, 6,391,592 and U.S.Ser. Nos. 09/916,135, 09/920,491 (U.S. Patent Application Publication20030096235), U.S. Ser. No. 09/910,292 (U.S. Patent ApplicationPublication 20030082543), and U.S. Ser. No. 10/013,598.

Methods for conducting polynucleotide hybridization assays have beenwell developed in the art. Hybridization assay procedures and conditionswill vary depending on the application and are selected in accordancewith the general binding methods known including those referred to in:Maniatis et al. Molecular Cloning: A Laboratory Manual (2^(nd) Ed. ColdSpring Harbor, N.Y, 1989); Berger and Kimmel Methods in Enzymology, Vol.152, Guide to Molecular Cloning Techniques (Academic Press, Inc., SanDiego, Calif., 1987); Young and Davism, P.N.A.S, 80: 1194 (1983).Methods and apparatus for carrying out repeated and controlledhybridization reactions have been described in U.S. Pat. Nos. 5,871,928,5,874,219, 6,045,996 and 6,386,749, 6,391,623 each of which areincorporated herein by reference

The present invention also contemplates signal detection ofhybridization between ligands in certain preferred embodiments. See U.S.Pat. Nos. 5,143,854, 5,578,832; 5,631,734; 5,834,758; 5,936,324;5,981,956; 6,025,601; 6,141,096; 6,185,030; 6,201,639; 6,218,803; and6,225,625, in U.S. Ser. No. 10/389,194 and in PCT ApplicationPCT/US99/06097 (published as WO99/47964), each of which also is herebyincorporated by reference in its entirety for all purposes.

Methods and apparatus for signal detection and processing of intensitydata are disclosed in, for example, U.S. Pat. Nos. 5,143,854, 5,547,839,5,578,832, 5,631,734, 5,800,992, 5,834,758; 5,856,092, 5,902,723,5,936,324, 5,981,956, 6,025,601, 6,090,555, 6,141,096, 6,185,030,6,201,639; 6,218,803; and 6,225,625, in U.S. Ser. Nos. 10/389,194,60/493,495 and in PCT Application PCT/US99/06097 (published asWO99/47964), each of which also is hereby incorporated by reference inits entirety for all purposes.

The practice of the present invention may also employ conventionalbiology methods, software and systems. Computer software products of theinvention typically include computer readable medium havingcomputer-executable instructions for performing the logic steps of themethod of the invention. Suitable computer readable medium includefloppy disk, CD-ROM/DVD/DVD-ROM, hard-disk drive, flash memory, ROM/RAM,magnetic tapes and etc. The computer executable instructions may bewritten in a suitable computer language or combination of severallanguages. Basic computational biology methods are described in, forexample Setubal and Meidanis et al., Introduction to ComputationalBiology Methods (PWS Publishing Company, Boston, 1997); Salzberg,Searles, Kasif, (Ed.), Computational Methods in Molecular Biology,(Elsevier, Amsterdam, 1998); Rashidi and Buehler, Bioinformatics Basics:Application in Biological Science and Medicine (CRC Press, London, 2000)and Ouelette and Bzevanis Bioinformatics: A Practical Guide for Analysisof Gene and Proteins (Wiley & Sons, Inc., 2^(nd) ed., 2001). See U.S.Pat. No. 6,420,108.

The present invention may also make use of various computer programproducts and software for a variety of purposes, such as probe design,management of data, analysis, and instrument operation. See, U.S. Pat.Nos. 5,593,839, 5,795,716, 5,733,729, 5,974,164, 6,066,454, 6,090,555,6,185,561, 6,188,783, 6,223,127, 6,229,911 and 6,308,170.

Additionally, the present invention may have preferred embodiments thatinclude methods for providing genetic information over networks such asthe Internet as shown in U.S. Ser. Nos. 10/197,621, 10/063,559 (UnitedStates Publication No. 20020183936), U.S. Ser. No. 10/065,856,10/065,868, 10/328,818, 10/328,872, 10/423,403, and 60/482,389.

b) Definitions

An “array” represents an intentionally created collection of moleculeswhich can be prepared either synthetically or biosynthetically. Inparticular, the term “array” herein means an intentionally createdcollection of probes (as used herein typically polymers, peptides,polynucleotides and or oligonucleotides) attached to at least a firstsurface of at least one solid support wherein the identity of eachpolynucleotide at a given predefined region or positionally definedlocation is known. The terms “array,” “biological chip” and “chip” areused interchangeably. A polymer array also can include only a subset ofthe complete set of probes. Similarly, a given array can exist on morethan one separate substrate, e.g., where the number of sequencesnecessitates a larger surface area or more than one solid substrate inorder to include all of the desired oligonucleotide sequences.

“Solid support,” “support,” and “substrate” refer to a material or groupof materials having a rigid or semi-rigid surface or surfaces. In manyembodiments, at least one surface of the solid support will besubstantially flat, although in some embodiments it may be desirable tophysically separate synthesis regions for different compounds with, forexample, wells, raised regions, pins, etched trenches, or the like.According to other embodiments, the solid support(s) will take the formof beads, resins, gels, microspheres, fibers or other geometricconfigurations.

The phrase “coupled to a support” means bound directly or indirectlythereto including attachment by covalent binding, hydrogen bonding,ionic interaction, hydrophobic interaction, or otherwise.

“Nucleotide” and “ribonucleotide” refers to both naturally occurring andnon-naturally occurring compounds having a heterocyclic base, a sugar,and a linking group, preferably a phosphate ester. For example,structural groups may be added to the ribosyl or deoxyribosyl unit ofthe nucleotide, such as a methyl or allyl group at the 2′-O position ora fluoro group that substitutes for the 2′-O group. The linking group,such as a phosphodiester, of the nucleic acid may be substituted ormodified, for example with methyl phosphonates or O-methyl phosphates.Bases and sugars can also be modified, as is known in the art. Forexample, unless otherwise limited the phrase would also cover syntheticand naturally occurring variants of nucleic acids, including withoutlimitation, base variants such as 7-deazapurine, 8-aza-7-deazapurine,isocytosine, pseudo isocytosine, and isouracil.

The terms “nucleic acid” or “nucleic acid molecule” as used herein referto a deoxyribonucleotide or ribonucleotide (see above) polymer in eithersingle or double stranded form. These terms also encompass DNA-RNAhybrids.

The term “sugar” as used herein relates to monosaccharide moieties.Preferred sugars are in cyclic form, for example, in furanose(5-membered ring) or pyranose (6-membered ring) forms. Sugars may be inany of their enantiomeric, diasteriomeric or stereoisomeric forms.

As used herein, the terms “oligonucleotide” and “polynucleotide” areused interchangeably in the conventional sense to refer to moleculescomprising two or more nucleosides, each nucleoside being linked to atleast one other nucleoside by an internucleoside linkage. Theoligonucleotides of the present invention may be linear, branched, orcyclic, but are preferably linear.

The term “mature oligonucleotide” or “full length oligonucleotide”refers to an oligonucleotide which has successfully undergone each ofits coupling reactions so that its sequence is complete and as expectedper the synthetic scheme.

The term “prematurely terminated” or “truncated” refers to anoligonucleotide which has failed at one or more coupling reactions sothat its sequence lacks specific units intended per the syntheticscheme.

The term “reactive functional groups” refers to functional groups whichmay react with other available functional groups under specifiedconditions to yield a covalent linkage. Examples of preferred reactivefunctional groups are hydroxyl (i.e., —OH) and phosphoramidite (i.e.,—OP(OR′)NR₂ wherein R′ and R are organic groups comprising 1 to 20carbon atoms). A preferred group of phosphoramidite functional groupsare those for which R′ is —CH₃, —CH₂CH₃, —CH₂CH₂CN, or —C₆H₄Cl; and R is—CH(CH₃)₂.

The term “protected” or “otherwise unreactive” functional groups refersto functional groups which are essentially unreactive toward otheravailable functional groups under specified conditions. The term“functional group protection” is used herein in the conventionalchemical sense to refer to common chemical methods employed toreversibly render unreactive a functional group, which otherwise wouldbe reactive, under specified conditions (such as pH, temperature,radiation, solvent, and the like). A wide variety of such “protecting”,“blocking”, or “masking” methods are widely used and well known inorganic synthesis. For example, a compound which has two non-equivalentreactive functional groups, both of which would be reactive underspecified conditions, may be derivatized to render one of the functionalgroups “protected”, and therefore unreactive, under the specifiedconditions; so protected, the compound may be used as a reactant whichhas effectively only one reactive functional group. After the desiredreaction (involving the reactive functional group) is complete, theprotected group may be “deprotected” to return it to its originalfunctionality.

A wide variety of protecting group strategies are known. For example,hydroxyl groups (i e., —OH) which are reactive toward a certain otherfunctional groups (for example, phosphoramidite) under alkalineconditions might be “protected” by conversion to a suitable ether, whichis unreactive under alkaline conditions. When it is desired to“deprotect” the hydroxyl group, the protected compound might be treatedwith acid. For example, an —OH group may be protected by reaction withDMT-Cl to yield the acid-labile -ODMT group which may be deprotected,for example, by treatment with a suitable acid, such as dichloroaceticacid.

Methods of obtaining nucleic acid sequences of a given length and knownsequence are known to those of skill in the art. Methods of solid phaseoligonucleotide synthesis are described in, for example: Advances in theSynthesis of Oligonucleotides by the Phosphoramidite Approach, Beaucage,S. L.; Iyer, R. P., Tetrahedron, 1992, 48, 2223-2311; U.S. Pat. Nos.4,58,066; 4,500,707; 5,132,418; 4,973,679; 4,415,732; Re. 34,069; and5,026,838.

The term “linker” means a molecule or group of molecules attached to asubstrate and spacing a synthesized polymer from the substrate forexposure/binding to a receptor.

The term “activation energy wavelength” refers to that wavelength ofelectromagnetic radiation that will activate a photoprotective group orphotocleavable group.

The term “activator” refers to a compound that facilitates coupling ofone nucleic acid to another, preferably in 3′-position of one nucleicacid to 5′-position of the other nucleic acid or vice a versa.

The terms “quality,” “performance” and “intensity” are usedinterchangeably herein when referring to oligonucleotide probes orbinding of a target molecule to oligonucleotide probes mean sensitivityof oligonucleotide probes to bind to a target molecule while giving aminimum of false signals.

The term “wafer” generally refers to a substantially flat sample ofsubstrate (i.e., solid-support) from which a plurality of individualarrays or chips can be fabricated.

The term “functional group” means a reactive chemical moiety present ona given monomer, polymer, linker or substrate surface. Examples offunctional groups include, e.g., the 3′ and 5′ hydroxyl groups ofnucleotides and nucleosides, as well as the reactive groups on thenucleobases of the nucleic acid monomers, e.g., the exocyclic aminegroup of guanosine, as well as amino and carboxyl groups on amino acidmonomers.

The term photoprotecting group (also called photolabile protectinggroups or photogroup for short) means a material which is chemicallybound to a reactive functional group on a monomer unit, linker, orpolymer and which may be removed upon selective exposure toelectromagnetic radiation or light, especially ultraviolet and visiblelight.

The term “reactive group” refers to a group that allows a covalentreaction to occur between for example a monomer and a linker or betweena second monomer and a first attached monomer. A reactive group may beprotected by photoprotective removable group. Removal of the photogroup,yields a deprotected reactive group. The terms “array” and “chip” areused interchangeably herein and refer to the final product of theindividual array of nucleic acid or oligonucleotide sequences, having aplurality of positionally distinct oligonucleotide sequences coupled tothe surface of the substrate. “Array” is used with reference to nucleicacid or oligonucleotide, but it should be appreciated that either canbe-attached to a solid support. Reference will be made toolinonucleotide arrays as a preferred example of the present invention.

The term “alkyl” refers to a branched or straight chain acyclic,monovalent saturated hydrocarbon radical of one to twenty carbon atoms.The term “alkenyl” refers to an unsaturated hydrocarbon radical whichcontains at least one carbon-carbon double bond and includes straightchain, branched chain and cyclic radicals.

The term “alkynyl” refers to an unsaturated hydrocarbon radical whichcontains at least one carbon-carbon triple bond and includes straightchain, branched chain and cyclic radicals.

The term “aryl” refers to an aromatic monovalent carboxylic radicalhaving a single ring (e.g., phenyl) or two condensed rings (e.g.,naphthyl), which can optionally be mono-, di-, or tri-substituted,independently, with alkyl, lower-alkyl, cycloalkyl, ydroxylower-alkyl,aminoloweralkyl, hydroxyl, thiol, amino, halo, nitro, lower-alkylthio,lower-alkoxy, mono-lower-alkylamino, di-lower-alkylamino, acyl,hydroxycarbonyl, lower-alkoxycarbonyl, hydroxysulfonyl,lower-alkoxysulfonyl, lower-alkylsulfonyl, lower-alkylsulfinyl,trifluoromethyl, cyano, tetrazoyl, carbamoyl, lower-alkylcarbamoyl, anddi-lower-alkylcarbamoyl.

Alternatively, two adjacent positions of the aromatic ring may besubstituted with a methylenedioxy or ethylenedioxy group. The term“heteroaromatic” refers to an aromatic monovalent mono- or poly-cyclicradical having at least one heteroatom within the ring, e.g., nitrogen,oxygen or sulfur, wherein the aromatic ring can optionally be mono-, di-or tri-substituted, independently, with alkyl, lower-alkyl, cycloalkyl,hydroxylower-alkyl, aminolower-alkyl, hydroxyl, thiol, amino, halo,nitro, lower-alkylthio, loweralkoxy, mono-lower-alkylamino,di-lower-alkylamino, acyl, hydroxycarbonyl, lower-alkoxycarbonyl,hydroxysulfonyl, lower-alkoxysulfonyl, lower-alkylsulfonyl,lower-alkylsulfinyl, trifluoromethyl, cyano, tetrazoyl, carbamoyl,loweralkylcarbamoyl, and di-lower-alkylcarbamoyl. For example, typicalheteroaryl groups with one or more nitrogen atoms are tetrazoyl, pyridyl(e.g., 4-pyridyl, 3-pyridyl, 2-pyridyl), pyrrolyl (e.g., 2-pyrrolyl,2-(N-alkyl)pyrrolyl), pyridazinyl, quinolyl (e.g. 2-quinolyl, 3-quinolyletc.), imidazolyl, isoquinolyl, pyrazolyl, pyrazinyl, pyrimidinyl,pyridonyl or pyridazinonyl; typical oxygen heteroaryl radicals with anoxygen atom are 2-furyl, 3-furyl or benzofuranyl; typical sulfurheteroaryl radicals are thienyl, and benzothienyl; typical mixedheteroatom heteroaryl radicals are furazanyl and phenothiazinyl.

Further the term also includes instances where a heteroatom within thering has been oxidized, such as, for example, to form an N-oxide orsulfone. The term “optionally substituted” refers to the presence orlack thereof of a substituent on the group being defined. Whensubstitution is present the group may be mono-, di- or tri-substituted,independently, with alkyl, lower-alkyl, cycloalkyl, hydroxylower-alkyl,aminoloweralkyl, hydroxyl, thiol, amino, halo, nitro, lower-alkylthio,lower-alkoxy, mono-lower-alkylamino, di-lower-alkylamino, acyl,hydroxycarbonyl, lower-alkoxycarbonyl, hydroxysulfonyl,lower-alkoxysulfonyl, lower-alkylsulfonyl, lower-alkylsulfinyl,trifluoromethyl, cyano, tetrazoyl, carbamoyl, lower-alkylcarbamoyl, anddi-lower-alkylcarbamoyl. Typically, electron-donating substituents suchas alkyl, lower-alkyl, cycloalkyl, hydroxylower-alkyl, aminolower-alkyl, hydroxyl, thiol, amino, halo, lower-alkylthio, lower-alkoxy,mono-lower-alkylamino and di-lower-alkylamino are preferred.

The term “electron donating group” refers to a radical group that has alesser affinity for electrons than a hydrogen atom would if it occupiedthe same position in the molecule. For example, typical electrondonating groups are hydroxy, alkoxy (e.g. methoxy), amino, alkylaminoand dialkylamine.

The term “leaving group” means a group capable of being displaced by anucleophile in a chemical reaction, for example halo, nitrophenoxy,pentafluorophenoxy, alkyl sulfonates (e.g., methanesulfonate), arylsulfonates, phosphates, sulfonic acid, sulfonic acid salts, and thelike.

“Activating group” refers to those groups which, when attached to aparticular functional group or reactive site, render that site morereactive toward covalent bond formation with a second functional groupor reactive site. The group of activating groups which are useful for acarboxylic acid include simple ester groups and anhydrides. The estergroups include alkyl, aryl and alkenyl esters and in particular suchgroups as 4-nitrophenyl, N-hydroxylsuccinimide and pentafluorophenol.Other activating groups are known to those of skill in the art.

A “cleavable moiety” or “releasable group” refers to a molecule whichcan be cleaved or released under a set of distinct conditions, e.g.,certain wave lengths of light or certain chemical conditions. Asemployed in the context of the present invention of arrays of releasablepolymer the conditions much be such as not to substantially damage orharm the polymer in questions. Persons of skill in the art willrecognize what cleavable moiety may be employed for example where thepolymer is a nucleic acid or a peptide.

“Predefined region” refers to a localized area on a solid support. Itcan be where synthesis takes place or where a nucleic acid is placed.Predefined region can also be defined as a “selected region.” Thepredefined region may have any convenient shape, e.g., circular,rectangular, elliptical, wedge-shaped, etc. For the sake of brevityherein, “predefined regions” are sometimes referred to simply as“regions.” In some embodiments, a predefined region and, therefore, thearea upon which each distinct compound is synthesized or placed issmaller than about 1 cm2 or less than 1 mm2. Within these regions, themolecule therein is preferably in a substantially pure form. Inadditional embodiments, a predefined region can be achieved byphysically separating the regions (i.e., beads, resins, gels, etc.) intowells, trays, etc.

A “linker” is a molecule or group of molecules attached to a substrateand spacing a synthesized polymer from the substrate for exposurebindingto a receptor.

A “channel block” is a material having a plurality of grooves orrecessed regions on a surface thereof. The grooves or recessed regionsmay take on a variety of geometric configurations, including but notlimited to stripes, circles, serpentine paths, or the like. Channelblocks may be prepared in a variety of manners, including etchingsilicon blocks, molding or pressing polymers, etc.

A “monomer” is a member of the set of small molecules which can bejoined together to form a polymer. The set of monomers includes but isnot restricted to, for example, the set of common L-amino acids, the setof common D-amino acids, the set of synthetic amino acids, the set ofnucleotides and the set of pentoses and hexoses. As used herein, monomerrefers to any member of a basis set for synthesis of a polymer. Thus,monomers refers to dimmers, trimers, tetramers and higher units ofmolecules which can be joined to form a polymer. For example, dimmers ofthe 20 naturally occurring L-amino acids for a basis set of 400 monomersfor synthesis of polypeptides. Different basis sets of monomers may beused at successive steps in the synthesis of a polymer. Furthermore,each of the sets may include protected members which are modified aftersynthesis.

A “polymer” is composed of two or more joined monomers and includes forexample both linear and cyclic polymers of nucleic acids,polysaccharides, phospholipids, and peptides having either α-, 0-, ando-amino acids, hetero-polymers in which a known drug is covalently boundto any of the above, polyurethanes, polyesters, polycarbonates,polyureas, polyamides, polyethyleneimines, polyarylene sulfides,polysiloxanes, polyimides, polyacetates, or other polymers.

A “releasable group” is a moiety or chemical group which is labile,i.e., may be activated or cleaved, under a given set of conditions, butis stable under other sets of conditions.

The term “monomer” as used herein refers to a single unit of polymer,which can be linked with the same or other monomers to form a biopolymer(for example, a single amino acid or nucleotide with two linking groupsone or both of which may have removable protecting groups) or a singleunit which is not part of a biopolymer. Thus, for example, a nucleotideis a monomer within an oligonucleotide polymer, and an amino acid is amonomer within a protein or peptide polymer; antibodies, antibodyfragments, chromosomes, plasmids, mRNA, cRNA, tRNA etc., for example,are also polymers.

The term “biopolymer” or sometimes refer by “biological polymer” as usedherein is intended to mean repeating units of biological or chemicalmoieties. Representative biopolymers include, but are not limited to,nucleic acids, oligonucleotides, amino acids, proteins, peptides,hormones, oligosaccharides, lipids, glycolipids, lipopolysaccharides,phospholipids, synthetic analogues of the foregoing, including, but notlimited to, inverted nucleotides, peptide nucleic acids, Meta-DNA, andcombinations of the above. It is important to note that biopolymers andpolymers are not mutually exclusive. Proteins, enzymes, DNA,polyethylene, RNA, are all polymers as they are derived from a repeatingmonomer units. However, proteins, enzymes, DNA are all biopolymers asmany of them first appeared in nature. Sometimes, it is not easy toclassify something as a biopolymer or a polymer. For example, vastnumber of human made amino acid derivatives and nucleotide derivativeshave been created and polymerized. Some of these are based on naturalproducts, many more are not. At this point the distinction between thetwo can be somewhat semantical.

The term “biopolymer synthesis” as used herein is intended to encompassthe synthetic production, both in situ (in the cell) and synthetically,e.g. by organic synthetic techniques outside of the cell, of abiopolymer. Related to a bioploymer is a “biomonomer”.

The term “combinatorial synthesis strategy” as used herein refers to acombinatorial synthesis strategy is an ordered strategy for parallelsynthesis of diverse polymer sequences by sequential addition ofreagents which may be represented by a reactant matrix and a switchmatrix, the product of which is a product matrix. A reactant matrix is a1 column by m row matrix of the building blocks to be added. The switchmatrix is all or a subset of the binary numbers, preferably ordered,between 1 and m arranged in columns. A “binary strategy” is one in whichat least two successive steps illuminate a portion, often half, of aregion of interest on the substrate. In a binary synthesis strategy, allpossible compounds which can be formed from an ordered set of reactantsare formed. In most preferred embodiments, binary synthesis refers to asynthesis strategy which also factors a previous addition step. Forexample, a strategy in which a switch matrix for a masking strategyhalves regions that were previously illuminated, illuminating about halfof the previously illuminated region and protecting the remaining half(while also protecting about half of previously protected regions andilluminating about half of previously protected regions). It will berecognized that binary rounds may be interspersed with non-binary roundsand that only a portion of a substrate may be subjected to a binaryscheme. A combinatorial “masking” strategy is a synthesis which useslight or other spatially selective deprotecting or activating agents toremove protecting groups from materials for addition of other materialssuch as amino acids.

The term “complementary” as used herein refers to the hybridization orbase pairing between nucleotides or nucleic acids, such as, forinstance, between the two strands of a double stranded DNA molecule orbetween an oligonucleotide primer and a primer binding site on a singlestranded nucleic acid to be sequenced or amplified. Complementarynucleotides are, generally, A and T (or A and U), or C and G. Two singlestranded RNA or DNA molecules are said to be complementary when thenucleotides of one strand, optimally aligned and compared and withappropriate nucleotide insertions or deletions, pair with at least about80% of the nucleotides of the other strand, usually at least about 90%to 95%, and more preferably from about 98 to 100%. Alternatively,complementarity exists when an RNA or DNA strand will hybridize underselective hybridization conditions to its complement. Typically,selective hybridization will occur when there is at least about 65%complementary over a stretch of at least 14 to 25 nucleotides,preferably at least about 75%, more preferably at least about 90%complementary. See, M. Kanehisa Nucleic Acids Res. 12:203 (1984),incorporated herein by reference.

The term “copolymer” refers to a polymer that is composed of more thanone monomer. Copolymers may be prepared by polymerizing one or moremonomers to provide a copolymer.

The term “detectable moiety” means a chemical group that provides asignal. The signal is detectable by any suitable means, includingspectroscopic, photochemical, biochemical, immunochemical, electrical,optical or chemical means. In certain cases, the signal is detectable by2 or more means.

The detectable moiety provides the signal either directly or indirectly.A direct signal is produced where the labeling group spontaneously emitsa signal, or generates a signal upon the introduction of a suitablestimulus. Radiolabels, such as ³H, ¹²⁵I, ³⁵S, ¹⁴C or ³²P, and magneticparticles, such as Dynabeads™, are nonlimiting examples of groups thatdirectly and spontaneously provide a signal. Labeling groups thatdirectly provide a signal in the presence of a stimulus include thefollowing nonlimiting examples: colloidal gold (40-80 nm diameter),which scatters green light with high efficiency; fluorescent labels,such as fluorescein, Texas red, Rhoda mine, and green fluorescentprotein (Molecular Probes, Eugene, Oreg.), which absorb and subsequentlyemit light; chemiluminescent or bioluminescent labels, such as luminol,lophine, acridine salts and luciferins, which are electronically excitedas the result of a chemical or biological reaction and subsequently emitlight; spin labels, such as vanadium, copper, iron, manganese andnitroxide free radicals, which are detected by electron spin resonance(ESR) spectroscopy; dyes, such as quinoline dyes, triarylmethane dyesand acridine dyes, which absorb specific wavelengths of light; andcolored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.)beads. See U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345;4,277,437; 4,275,149 and 4,366,241.

A detectable moiety provides an indirect signal where it interacts witha second compound that spontaneously emits a signal, or generates asignal upon the introduction of a suitable stimulus. Biotin, forexample, produces a signal by forming a conjugate with streptavidin,which is then detected. See Hybridization With Nucleic Acid Probes. InLaboratory Techniques in Biochemistry and Molecular Biology; Tijssen,P., Ed.; Elsevier: New York, 1993; Vol. 24. An enzyme, such ashorseradish peroxidase or alkaline phosphatase, that is attached to anantibody in a label-antibody-antibody as in an ELISA assay, alsoproduces an indirect signal.

A preferred detectable moiety is a fluorescent group. Fluorescent groupstypically produce a high signal to noise ratio, thereby providingincreased resolution and sensitivity in a detection procedure.Preferably, the fluorescent group absorbs light with a wavelength aboveabout 300 nm, more preferably above about 350 nm, and most preferablyabove about 400 nm. The wavelength of the light emitted by thefluorescent group is preferably above about 310 nm, more preferablyabove about 360 nm, and most preferably above about 410 nm.

The fluorescent detectable moiety is selected from a variety ofstructural classes, including the following nonlimiting examples: 1- and2-aminonaphthalene, p,p′diaminostilbenes, pyrenes, quaternaryphenanthridine salts, 9-aminoacridines, p,p′-diaminobenzophenone imines,anthracenes, oxacarbocyanine, marocyanine, 3-aminoequilenin, perylene,bisbenzoxazole, bis-p-oxazolyl benzene, 1,2-benzophenazin, retinol,bis-3-aminopridinium salts, hellebrigenin, tetracycline, sterophenol,benzimidazolyl phenylamine, 2-oxo-3-chromen, indole, xanthen,7-hydroxycoumarin, phenoxazine, salicylate, strophanthidin, porphyrins,triarylmethanes, flavin, xanthene dyes (e.g., fluorescein and rhodaminedyes); cyanine dyes; 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene dyes andfluorescent proteins (e.g., green fluorescent protein,phycobiliprotein).

A number of fluorescent compounds are suitable for incorporation intothe present invention. Nonlimiting examples of such compounds includethe following: dansyl chloride; fluoresceins, such as3,6-dihydroxy-9-phenylxanthhydrol; rhodamineisothiocyanate;N-phenyl-1-amino-8-sulfonatonaphthalene;N-phenyl-2-amino-6-sulfonatonaphthanlene;4-acetamido-4-isothiocyanatostilbene-2,2′-disulfonic acid;pyrene-3-sulfonic acid; 2-toluidinonapththalene-6-sulfonate; N-phenyl,N-methyl 2-aminonaphthalene-6-sulfonate; ethidium bromide; stebrine;auromine-0,2-(9′-anthroyl)palmitate; dansyl phosphatidylethanolamin;N,N′-dioctadecyl oxacarbocycanine; N,N′-dihexyl oxacarbocyanine;merocyanine, 4-(3′-pyrenyl)butryate; d-3-aminodesoxy-equilenin;12-(9′-anthroyl)stearate; 2-methylanthracene; 9-vinylanthracene;2,2′-(vinylene-p-phenylene)bisbenzoxazole; p-bis[2-(4-methyl-5-phenyloxazolyl)]benzene; 6-dimethylamino-1,2-benzophenzin; retinol;bis(3′-aminopyridinium)-1,10-decandiyl diiodide; sulfonaphthylhydrazoneof hellibrienin; chlorotetracycline;N-(7-dimethylamino-4-methyl-2-oxo-3-chromenyl)maleimide;N-[p-(2-benzimidazolyl)phenyl]maleimide; N-(4-fluoranthyl)maleimide;bis(homovanillic acid); resazarin;4-chloro-7-nitro-2,1,3-benzooxadizole; merocyanine 540; resorufin; rosebengal and 2,4-diphenyl-3(2H)-furanone. Preferably, the fluorescentdetectable moiety is a fluorescein or rhodamine dye.

Another preferred detectable moiety is colloidal gold. The colloidalgold particle is typically 40 to 80 nm in diameter. The colloidal goldmay be attached to a labeling compound in a variety of ways. In oneembodiment, the linker moiety of the nucleic acid labeling compoundterminates in a thiol group (—SH), and the thiol group is directly boundto colloidal gold through a dative bond. See Mirkin et al. Nature 1996,382, 607-609. In another embodiment, it is attached indirectly, forinstance through the interaction between colloidal gold conjugates ofantibiotin and a biotinylated labeling compound. The detection of thegold labeled compound may be enhanced through the use of a silverenhancement method. See Danscher et al. J. Histotech 1993, 16, 201-207.

The term “effective amount” as used herein refers to an amountsufficient to induce a desired result.

Although generally used herein to define separate regions containingdiffering polymer sequences, the term “feature” generally refers to anyelement, e.g., region, structure or the like, on the surface of asubstrate. Preferably, substrates will have small feature sizes, andconsequently, high feature densities on substrate surfaces. For example,individual features will typically have at least one of a length orwidth dimension that is no greater than 100 microns, and preferably, nogreater than 50 microns, and more preferably no greater than about 20microns. Preferred embodiments of the present invention may havefeatures as small as 1 micron, down to 0.5 microns. Thus, forembodiments employing substrates having a plurality of polymer sequenceson their surfaces, each different polymer sequence will typically besubstantially contained within a single feature.

The term “fragmentation” refers to the breaking of nucleic acidmolecules into smaller nucleic acid fragments. In certain embodiments,the size of the fragments generated during fragmentation can becontrolled such that the size of fragments is distributed about acertain predetermined nucleic acid length.

The term “genome” as used herein is all the genetic material in thechromosomes of an organism. DNA derived from the genetic material in thechromosomes of a particular organism is genomic DNA. A genomic libraryis a collection of clones made from a set of randomly generatedoverlapping DNA fragments representing the entire genome of an organism.

The term “hybridization” as used herein refers to the process in whichtwo single-stranded polynucleotides bind non-covalently to form a stabledouble-stranded polynucleotide; triple-stranded hybridization is alsotheoretically possible. The resulting (usually) double-strandedpolynucleotide is a “hybrid.” The proportion of the population ofpolynucleotides that forms stable hybrids is referred to herein as the“degree of hybridization.” Hybridizations are usually performed understringent conditions, for example, at a salt concentration of no morethan 1 M and a temperature of at least 25° C. For example, conditions of5×SSPE (750 mM NaCl, 50 mM NaPhosphate, 5 mM EDTA, pH 7.4) and atemperature of 25-30° C. are suitable for allele-specific probehybridizations. For stringent conditions, see, for example, Sambrook,Fritsche and Maniatis. “Molecular Cloning: A Laboratory Manual” 2^(nd)Ed. Cold Spring Harbor Press (1989) which is hereby incorporated byreference in its entirety for all purposes above.

The term “hybridization conditions” as used herein will typicallyinclude salt concentrations of less than about 1M, more usually lessthan about 500 mM and preferably less than about 200 mM. Hybridizationtemperatures can be as low as 5° C., but are typically greater than 22°C., more typically greater than about 30° C., and preferably in excessof about 37° C. Longer fragments may require higher hybridizationtemperatures for specific hybridization. As other factors may affect thestringency of hybridization, including base composition and length ofthe complementary strands, presence of organic solvents and extent ofbase mismatching, the combination of parameters is more important thanthe absolute measure of any one alone.

The term “hybridization probes” as used herein are oligonucleotidescapable of binding in a base-specific manner to a complementary strandof nucleic acid. Such probes include peptide nucleic acids, as describedin Nielsen et al., Science 254, 1497-1500 (1991), and other nucleic acidanalogs and nucleic acid mimetics.

The term “hybridizing specifically to” as used herein refers to thebinding, duplexing, or hybridizing of a molecule only to a particularnucleotide sequence or sequences under stringent conditions when thatsequence is present in a complex mixture (for example, total cellular)DNA or RNA.

The term “isolated nucleic acid” as used herein means an object speciesinvention that is the predominant species present (i.e., on a molarbasis it is more abundant than any other individual species in thecomposition). Preferably, an isolated nucleic acid comprises at leastabout 50, 80 or 90% (on a molar basis) of all macromolecular speciespresent. Most preferably, the object species is purified to essentialhomogeneity (contaminant species cannot be detected in the compositionby conventional detection methods).

The term “ligand” as used herein refers to a molecule that is recognizedby a particular receptor. The agent bound by or reacting with a receptoris called a “ligand,” a term which is definitionally meaningful only interms of its counterpart receptor. The term “ligand” does not imply anyparticular molecular size or other structural or compositional featureother than that the substance in question is capable of binding orotherwise interacting with the receptor. Also, a ligand may serve eitheras the natural ligand to which the receptor binds, or as a functionalanalogue that may act as an agonist or antagonist. Examples of ligandsthat can be investigated by this invention include, but are notrestricted to, agonists and antagonists for cell membrane receptors,toxins and venoms, viral epitopes, hormones (for example, opiates,steroids, etc.), hormone receptors, peptides, enzymes, enzymesubstrates, substrate analogs, transition state analogs, cofactors,drugs, proteins, and antibodies.

The term “mRNA,” or sometimes referred to as “mRNA transcripts,” as usedherein, includes, but not limited to pre-mRNA transcript(s), transcriptprocessing intermediates, mature mRNA(s) ready for translation andtranscripts of the gene or genes, or nucleic acids derived from the mRNAtranscript(s). Transcript processing may include splicing, editing anddegradation. As used herein, a nucleic acid derived from an mRNAtranscript refers to a nucleic acid for whose synthesis the mRNAtranscript or a subsequence thereof has ultimately served as a template.Thus, a cDNA reverse transcribed from an mRNA, an RNA transcribed fromthat cDNA, a DNA amplified from the cDNA, an RNA transcribed from theamplified DNA, etc., are all derived from the mRNA transcript anddetection of such derived products is indicative of the presence and/orabundance of the original transcript in a sample. Thus, mRNA derivedsamples include, but are not limited to, mRNA transcripts of the gene orgenes, cDNA reverse transcribed from the mRNA, cRNA transcribed from thecDNA, DNA amplified from the genes, RNA transcribed from amplified DNA,and the like.

The term “nucleic acid library” or sometimes refer by “array” as usedherein refers to an intentionally created collection of nucleic acidswhich can be prepared either synthetically or biosynthetically andscreened for biological activity in a variety of different formats (forexample, libraries of soluble molecules; and libraries of oligostethered to resin beads, silica chips, or other solid supports).Additionally, the term “array” is meant to include those libraries ofnucleic acids which can be prepared by spotting nucleic acids ofessentially any length (for example, from 1 to about 1000 nucleotidemonomers in length) onto a substrate. The term “nucleic acid” as usedherein refers to a polymeric form of nucleotides of any length, eitherribonucleotides, deoxyribonucleotides or peptide nucleic acids (PNAs),that comprise purine and pyrimidine bases, or other natural, chemicallyor biochemically modified, non-natural, or derivatized nucleotide bases.The backbone of the polynucleotide can comprise sugars and phosphategroups, as may typically be found in RNA or DNA, or modified orsubstituted sugar or phosphate groups. A polynucleotide may comprisemodified nucleotides, such as methylated nucleotides and nucleotideanalogs. The sequence of nucleotides may be interrupted bynon-nucleotide components. Thus the terms nucleoside, nucleotide,deoxynucleoside and deoxynucleotide generally include analogs such asthose described herein. These analogs are those molecules having somestructural features in common with a naturally occurring nucleoside ornucleotide such that when incorporated into a nucleic acid oroligonucleoside sequence, they allow hybridization with a naturallyoccurring nucleic acid sequence in solution. Typically, these analogsare derived from naturally occurring nucleosides and nucleotides byreplacing and/or modifying the base, the ribose or the phosphodiestermoiety. The changes can be tailor made to stabilize or destabilizehybrid formation or enhance the specificity of hybridization with acomplementary nucleic acid sequence as desired.

The term “nucleic acids” as used herein may include any polymer oroligomer of pyrimidine and purine bases, preferably cytosine, thymine,and uracil, and adenine and guanine, respectively. See Albert L.Lehninger, PRINCIPLES OF BIOCHEMISTRY, at 793-800 (Worth Pub. 1982).Indeed, the present invention contemplates any deoxyribonucleotide,ribonucleotide or peptide nucleic acid component, and any chemicalvariants thereof, such as methylated, hydroxymethylated or glucosylatedforms of these bases, and the like. The polymers or oligqmers may beheterogeneous or homogeneous in composition, and may be isolated fromnaturally-occurring sources or may be artificially or syntheticallyproduced. In addition, the nucleic acids may be DNA or RNA, or a mixturethereof, and may exist permanently or transitionally in single-strandedor double-stranded form, including homoduplex, heteroduplex, and hybridstates.

The term “polymorphism” as used herein refers to the occurrence of twoor more genetically determined alternative sequences or alleles in apopulation. A polymorphic marker or site is the locus at whichdivergence occurs. Preferred markers have at least two alleles, eachoccurring at frequency of greater than 1%, and more preferably greaterthan 10% or 20% of a selected population. A polymorphism may compriseone or more base changes, an insertion, a repeat, or a deletion. Apolymorphic locus may be as small as one base pair. Polymorphic markersinclude restriction fragment length polymorphisms, variable number oftandem repeats (VNTR's), hypervariable regions, minisatellites,dinucleotide repeats, trinucleotide repeats, tetranucleotide repeats,simple sequence repeats, and insertion elements such as Alu. The firstidentified allelic form is arbitrarily designated as the reference formand other allelic forms are designated as alternative or variantalleles. The allelic form occurring most frequently in a selectedpopulation is sometimes referred to as the wildtype form. Diploidorganisms may be homozygous or heterozygous for allelic forms. Adiallelic polymorphism has two forms. A triallelic polymorphism hasthree forms. Single nucleotide polymorphisms (SNPs) are included inpolymorphisms.

The term “primer” as used herein refers to a single-strandedoligonucleotide capable of acting as a point of initiation fortemplate-directed DNA synthesis under suitable conditions for example,buffer and temperature, in the presence of four different nucleosidetriphosphates and an agent for polymerization, such as, for example, DNAor RNA polymerase or reverse transcriptase. The length of the primer, inany given case, depends on, for example, the intended use of the primer,and generally ranges from 15 to 30 nucleotides. However, longer primersare also preferred including from 80 to 160 nucleotides. Short primermolecules generally require cooler temperatures to form sufficientlystable hybrid complexes with the template. A primer need not reflect theexact sequence of the template but must be sufficiently complementary tohybridize with such template. The primer site is the area of thetemplate to which a primer hybridizes. The primer pair is a set ofprimers including a 5′ upstream primer that hybridizes with the 5′ endof the sequence to be amplified and a 3′ downstream primer thathybridizes with the complement of the 3′ end of the sequence to beamplified.

The term “probe” is often used in the context of array technology as thepolymer bound to the array. See U.S. Pat. No. 6,582,908 for an exampleof arrays having all possible combinations of probes with 10, 12, andmore bases. Examples of probes that can be investigated by thisinvention include, but are not restricted to, agonists and antagonistsfor cell membrane receptors, toxins and venoms, viral epitopes, hormones(for example, opioid peptides, steroids, etc.), hormone receptors,peptides, enzymes, enzyme substrates, cofactors, drugs, lectins, sugars,oligonucleotides, nucleic acids, oligosaccharides, proteins, andmonoclonal antibodies. As used herein, probes may be designed to bereleasable, i.e., capable of being severed from the array and, thus, asinterchangeable “primers.”

c) Massively Parallel Oligonucleotide Probe Synthesis and Purification

According to one aspect of the present invention, massively paralleloligonucleotide probe synthesis and purification is provided forapplications that utilize large collections of defined high-fidelityoligonucleotides (e.g., from about 10¹ to about 10⁵ different sequences,generally between 25-160 bases in length) with or without authentic3′-hydroxy termini. Preferably, the oligonucleotides are between about80 to 160. It is also preferred that the oligonucleotides containauthentic 3′ hydroxyl groups. However, other types of 3′ hydroxyl groupsare also preferred, such as phosphorylated.

In accordance with one aspect of the present invention,photolithographic synthetic strategies may provide a convenient approachto efficiently produce an array of such probes. In accordance with anaspect of the present invention, synthesis may be initiated with areverse-orientation RNA monomer that contains an orthogonal 2′-OH groupwith an appropriate protective group. Two exemplary monomers are shownin FIG. 2. Following conventional 3′→5′ oligonucleotide synthesis, the2′-OH protecting group is removed to allow base-induced intramoleculartransesterification. The transesterification reaction causes release ofthe synthesized oligonucleotide, leaving an authentic 3′-hydroxyfunctionality, while the 2′,3′-cyclic phosphate remains attached to thesolid support (see FIG. 1).

Accordingly, one embodiment of the present invention provides a methodof fabricating a plurality of oligonucleotides having free 3′-hydroxylgroups from a high density oligonucleotide array, said method comprisingthe steps of

-   a) providing a solid substrate comprising a plurality of    ribonucleotides attached thereto at a density, one said    ribonucleotide shown below

wherein PG₁ is protecting group 1, PG₂ is protecting group 2, B is anaturally or non-naturally occurring base, and said ribonucleotide isattached to said substrate through the 5′-hydroxyl group;

-   b) selectively removing PG₁ in pre-selected areas of the substrate    to provide a plurality of free 3′-hydroxyl groups on said    ribonucloetide;-   c) reacting said free 3′-hydroxyl groups with a    2′-deoxyribonucleotide having the structure

wherein PG₃ is protecting group 3 and RG is a reactive group to couplesaid 2′-deoxyribonucleotide to said ribonucleotide to provide thestructure

-   d) selectively removing PG₃ from the 5′-hydroxyl of said    2′-deoxyribonucleotide in pre-selected areas to provide a plurality    of free 5′-hydroxyl groups;-   e) reacting said free 5′-hydroxyl groups with an additional    2′-deoxyribonucleotide having the structure

to yield a product of the structure

-   f) repeating steps d and e one or more times to provide said    oligonucleotides attached to said solid substrate;-   g) removing PG₂ from one or more of said ribonucleotides to provide    a free 2′-hydroxyl group on each of said one or more    ribonucleotides; and-   h) transesterifying each of said one or more ribonucleotides to    yield said solid substrate having a cyclic ester attached thereto    and free oligonucleotides, each oligonucleotide having a 3′-hydroxyl    group and having the structure

In certain aspects of the present invention the 2′-deoxyribonucleotidehas a phosphoramidite reactive group as shown by

wherein R₁ is selected from cyanoethyl, methyl, t-butyl, trimethylsilylor the like, and R₂ and R₃ are independently selected from isopropyl,cyclohexyl or the like.

In certain aspects of the present invention the oligonucleotides areprobes while in other aspects of the present invention theoligonucleotides are primers.

In one aspect of the present invention, PG₁, PG₃ and PG₄ areindependently selected protecting groups. These protecting groups may bethe same as or different from one another. It has been discovered inaccordance with the present invention that to achieve suitable primerpurity and quantity, a highly-efficient photogroup (>90% averagestepwise coupling efficiency) is preferred for PG₁, PG₃ and PG₄, such asNPPOC or MBPMOC:

Both NNPOC and MBPMOC give greater than 90% stepwise coupling. Forexample NNPOC gives 97-98% stepwise coupling.

Alternatively, DMT-based chemistry could be employed in conjunction withphotoacid. DMT-based resist methodologies have been found, in accordancewith the present invention to provide up to a 99% stepwise yield. Incertain embodiments of the present invention, when PG₁, PG₃ or PG₄ areDMT, the DMT group may be removed in selected areas by exposure to acidgenerated by a photoacid generator in the presence of electro magneticradiation of an appropriate wavelength in the presence of an acidscavenger. In further embodiments of the present invention the acidscavenger is selected from the group consisting of organic bases andpolymeric bases. In preferred aspects of the present invention, the acidscavenger is a polymeric base.

In another aspect of the present invention, it is further contemplatedthat combinations of DMT-based and photochemical-based probe assemblycould be performed, for example to assemble common regions of the probesequence. Despite the specific primer array synthesis methodology,high-density substrates (200-2000 pmoles/cm²) can be employed tosignificantly boost primer yield. Such substrates are typically basedupon three-dimensional architectures, thin-films or polymeric coatings.

The rate of transesterification (i.e., oligonucleotide release) can besignificantly enhanced by raising the pH of the aqueous solution (pH9-12) and/or by the addition of particular metal ions, which are knownin the art to facilitate or catalyze such reactions.

Exemplary 2′-OH RNA monomer protecting groups, PG₂, are Ac (removedduring base deprotection), FPMP or CEE (removed with mild acid, but notstrong acid), TBDMS or TOM (removed with fluoride ions) or even aphotogroup that is active at wavelengths longer than 365 nm.

In preferred embodiments of the present invention PG₂ will be orthogonalto PG₁, PG₃, and PG₄. As such, the conditions used to deprotect PG₂ aredifferent than those used to deprotect PG₁, PG₃ and PG₄. For example, ifacid labile protecting groups are used for PG₁, PG₃ and PG₄, then PG₂may be protected with a photolabile protecting group. The converse isalso true, i.e., if photolabile protecting groups are used for PG₁, PG₃and PG₄, then PG₂ may be protected with an acid labile protecting group.In some embodiments, photo labile protecting groups can be used for allof PG₁, PG₂, PG₃ and PG₄, provided that the photo labile protectinggroup of PG₂ is active at wavelengths longer than those of the photolabile protecting group of PG₁, PG₃ and PG₄, e.g., longer than 365 nm.

Because the coupling reactions between the free 5′-hydroxyl groups andthe reactive group of the 2′-deoxyribonucleotide typically are not 100%efficient in a finite time period, a small percentage of truncatedsequences, i.e., sequences which did not undergo the expected couplingevent, is produced at each coupling step. To prevent these truncatedsequences from undergoing further coupling reactions to produceunexpected oligonucleotide sequences, the unreacted free 5′-hydroxylgroup can be capped prior to performing additional coupling reactions torender it unreactive for subsequent synthesis steps. This cappingreaction may be accomplished by acetylation using methods known in theart.

In another aspect of the present invention, steps are employed to enrichor purify the full-length oligonucleotides (e.g., probes or primers).For example, if a base-stable RNA monomer is employed, the final 5′-DMTgroup is retained on the probe and the array is deprotected in the usualmanner. The deprotection step removes the DNA protecting groups,reverses some unwanted chemical modifications, and also cleavesdepurinated sites. The protecting groups and truncated fragments arewashed away from the solid support, leaving behind the immobilizedfull-length probes that contain a 5′-DMT group as well as immobilizedtruncated species that do not possess a 5′-DMT group.

A subsequent processing step causes removal of the 2′-OH RNA protectinggroup and subsequent transesterification/release of probes (FIG. 3). Thesolution containing the mixture of free oligonucleotides can then beadsorbed onto a disposable hydrophobic oligonucleotide purificationcartridge (e.g., Waters Sep-Pak, ABI OPC, etc.) to isolate only thoseoligonucleotide species that possess a 5′-DMT group.

Accordingly, the present invention provides a method of purifying a setof oligonucleotides, comprising the steps of

-   a) providing a solid substrate comprising a plurality of    ribonucleotides attached thereto at a density, one said    ribonucleotide shown below

wherein PG₁ is protecting group 1, PG₂ is an alkalai resistantprotecting group, B is a naturally or non-naturally occurring base, andsaid ribonucleotide is attached to said substrate through the5′-hydroxyl group;

-   b) selectively removing PG₁ in pre-selected areas of the substrate    to provide a plurality of free 3′-hydroxyl groups on said    ribonucloetide;-   c) reacting said free 3′-hydroxyl groups with a    2′-deoxyribonucleotide having the structure

wherein PG₃ is DMT, B is a naturally or non-naturally occurring base inwhich the exocyclic amine groups are protected with alkalai labileprotecting groups, and RG is a reactive group to couple said2′-deoxyribonucleotide to said ribonucleotide to provide the structure

-   d) selectively removing PG₃ from the 5′-hydroxyl of said    2′-deoxyribonucleotide in pre-selected areas to provide a plurality    of free 5′-hydroxyl groups;-   e) reacting said free 5′-hydroxyl groups with an additional    2′-deoxyribonucleotide having the structure

wherein PG₄ is DMT, to yield a product of the structure

-   f) repeating steps d and e one or more times to provide said    oligonucleotides attached to said solid substrate;-   g) deprotecting said set of oligonucleotides while said    oligonucleotides are still attached to said substrate by subjecting    said oligonucleotides to alkaline conditions, wherein said alkaline    conditions remove said alkalai labile protecting groups acting to    protect said exocyclic amines and in addition cleave depurinated    DNA, leaving a 3′-end of the cleaved depurinated strand attached to    the substrate and releasing a truncated fragment;-   h) washing the solid support to remove said released truncated    fragments and protecting groups, leaving full length    oligonucleotides having a DMT group on the 5′-hydroxyl group and    truncated oligonucleotides without the 5′-DMT group;-   i) removing PG₂ from one or more of said ribonucleotides to provide    a free 2′-hydroxyl group on each of said one or more    ribonucleotides;-   j) transesterifying each of said one or more ribonucleotides to    yield said solid substrate having a cyclic ester attached thereto    and a mixture of full length oligonucleotides having 5′-DMT groups    and free 3′-hydroxyl groups and having the structure

and truncated fragments lacking the DMT group;

-   k) applying the mixture to hydrophobic oligonucleotide purification    resin to isolate only those oligonucleotides having the 5′-DMT group    to yield full length oligonucleotides; and-   l) removing the 5′-DMT group to provide full length oligonucleotides    having both 5′- and 3′-hydroxyl groups.

As an alternative to the procedure described above, the 2′-OH RNAprotecting group is simply Ac, which is removed along with the otherprotecting groups during standard deprotection. If the non-catalyzedtransesterification rate is low, then full-length oligonucleotides willnot be substantially released until the reaction conditions areappropriately adjusted to cause transesterification.

It is contemplated that reporter groups (e.g., chromophores,fluoraphores, detectable labels) or affinity tags (e.g., biotin) can beincorporated into the probe sequences, in either single-color ormulti-color formats. Phosphorylation at either terminus (or bothtermini) is also possible. Dual-labeled oligonucleotide “probes” (e.g.,TaqMan probes and molecular beacons) are also contemplated in accordancewith the instant invention. Additionally, non-conventional buildingblocks (e.g., nucleoside analogues or mimics) could be incorporated intothe probe/primer, either in part or in whole. Primer quantity will be afunction of the stepwise coupling yield, primer length, the surfaceloading, feature size and feature redundancy of a given array design.The relative concentration of each probe/primer can be adjusted bycontrolling the redundancy of the array design.

It should be noted that many of the embodiments described herein aredescribed with reference to the fabrication of oligonucleotide probesand/or primers. However, these descriptive embodiments are not intendedto limit the types of or uses for oligonucleotides which can be producedand purified or isolated using the described methods. Theoligonucleotides produced by the described methods can have any desiredsequence composition and be utilized in any context in whicholigonucleotides of defined sequence are desired. Oligonucleotides canbe prepared according to the methods of the invention singly or in setswithin the larger population of distinct sequences. By way ofnon-limiting example, forward and reverse PCR primers for a particulartarget sequence may be synthesized as a set on the array; alternatively,sets of probes or primers differing by a single base at specificpositions can be produced.

A preferred aspect of the present invention is that synthesis isinitiated with a reverse-orientation RNA monomer that contains anorthogonal 2′-OH protecting group. Following conventional 3′→5′ probesynthesis, the 2′-OH protecting group is removed to allow base-inducedintramolecular transesterification. The transesterification reactioncauses release of the synthesized probe with an authentic 3′-hydroxyfunctionality, while the 2′,3′-cyclic phosphate remains attached to thesolid support (see FIG. 1). The rate of transesterification (i.e., proberelease) can be significantly enhanced by raising the pH of the aqueoussolution (pH 9-12) and/or by the addition of particular metal ions,which are known in the art to facilitate or catalyze such reactions.

Two exemplary monomers are depicted in FIG. 2: Exemplary 2′-OH RNAmonomer protecting groups are Ac (removed during base deprotection),FPMP or CEE (removed with mild acid, but not strong acid), TBDMS or TOM(removed with fluoride ions) or even a photogroup that is active atwavelengths longer than 365 nm.

Another preferred aspect of the present invention is the steps that areemployed to enrich or purify the full-length probes (see FIG. 3). Forexample, if a base-stable RNA monomer is employed, the final 5′-DMTgroup is retained on the probe and the array is deprotected in the usualmanner. The deprotection step removes the DNA protecting groups,reverses some unwanted chemical modifications, and also cleavesdepurinated sites. The protecting groups and truncated fragments arewashed away from the solid support, leaving behind the immobilizedfull-length probes that contain a 5′-DMT group as well as truncatedspecies that do not possess a 5′-DMT group. A subsequent processing stepcauses removal of the 2′-OH RNA protecting group and subsequenttransesterification/release of probes. The solution containing themixture of probes can then be adsorbed onto a disposable hydrophobicoligonucleotide purification cartridge (e.g., Waters Sep-Pak, ABI OPC,etc.) to isolate only those probe species that possess a 5′-DMT group.

As an alternative to the procedure described above, the 2′-OH RNAprotecting group is simply Ac, which is removed along with the otherprotecting groups during standard deprotection. If the non-catalyzedtransesterification rate is low, then full-length probes will not besubstantially released until the reaction conditions are appropriatelyadjusted to cause transesterification.

It is contemplated that reporter groups (e.g., cluomophores,fluorophores, detectable labels) or affinity tags (e.g.,biotin) can beincorporated into the probe sequences, in either single-color ormulti-color formats. Phosphorylation at either terminus (or bothtermini) is also possible. Dual-labeled oligonucleotide “probes” (e.g.,TaqMan probes and molecular beacons) are also contemplated.Additionally, non-conventional building blocks (e.g., nucleosideanalogues or mimics) could be incorporated into the probe/primer, eitherin part or in whole. Primer quantity will be a function of the stepwisecoupling yield, primer length, the surface loading, feature size andfeature redundancy of a given array design. The relative concentrationof each primer can be adjusted by controlling the redundancy of thearray design.

In accordance with an aspect of the present invention, a method formassively parallel oligonucleotide probe synthesis and release of saidoligonucleotides from an array of probes on a solid substrate isprovided, the method having the steps of providing a solid substrate;

-   attaching a plurality of linkers to the substrate, each said linker    comprising a cleavable moiety, wherein said cleavable moiety is    activatable only at a distinct set of conditions and wherein    activation of said cleavable moiety disrupts the linker to allow of    a polymer bound to said linker, to provide a substrate with a    plurality of attached linkers;-   attaching a first monomer to at least one of said plurality of    attached linkers to provide an attached first monomer;-   attaching a second monomer to a least one of said attached first    monomers or said plurality of attached linkers to provide an    attached second monomer;-   attaching a third monomer to a least one of said attached first    monomer, said second monomer or said plurality of attached linkers    to provide an attached third monomer;-   repeating said steps of attaching monomers until the desired array    of polymers is complete;-   and subjecting the array to the distinct set of conditions to    release polymers from said array.

In a preferred embodiment of the present invention, the desired arrayhas between 10¹ to 10⁵ oligonucleotides of different sequences which arebetween about 80-160 bases in length.

According to another aspect of the present invention, it is preferredthat said released oligonucleotides have authentic 3′-hydroxy terminiupon exposure to said distinct set of conditions or are furtherprocessed to have authentic 3′ hydroxyl termini.

According to another aspect of the present invention, it is preferredthat the method further comprising the use of the photoprotectivegroups, preferably NPPOC or NNPOC to provide suitable primer purity andquantity.

According to another aspect of the present invention, it is preferredthat the method employ DMT-based photoresist groups to provide suitableprimer purity and quantity.

According to another aspect of the present invention, it is preferredthat ink-jet based in situ oligonucleotide synthesis is used to providethe oligonucleotide probes.

According to another aspect of the present invention, it is preferredthat the method is initiated with a reverse-orientation RNA monomer thatcontains an orthogonal 2′-OH protecting group.

According to another aspect of the present invention, it is preferredthat following conventional 3′→5′ probe synthesis, the 2′-OH protectinggroup is removed to allow base-induced intramoleculartransesterification. Base-induced intramolecular transesterification ispreferentially performed by raising the pH of the aqueous solution to pH9-12 or by the addition of particular metal ions.

All patents, patent applications, and literature cited in thespecification are hereby incorporated by reference in their entirety. Inthe case of any inconsistencies, the present disclosure, including anydefinitions therein will prevail.

The invention has been described with reference to various specific andpreferred embodiments and techniques. However, it should be understoodthat many variations and modifications may be made while remainingwithin the spirit and scope of the invention.

1. A method of fabricating a plurality of oligonucleotides having free3′-hydroxyl groups from a high density oligonucleotide array, saidmethod comprising the steps of a) providing a solid substrate comprisinga plurality of ribonucleotides attached thereto, one said ribonucleotideshown below

wherein PG₁ is protecting group 1, PG₂ is protecting group 2, B is anaturally or non-naturally occurring base, and said ribonucleotide isattached to said substrate through the 5′-hydroxyl group; b) selectivelyremoving PG₁ in pre-selected areas to provide a plurality of free3′-hydroxyl groups on said ribonucloetide; c) reacting said free3′-hydroxyl groups with a 2′-deoxyribonucleotide having the structure

wherein PG₃ is protecting group 3 and RG is a reactive group to couplesaid 2′-deoxyribonucleotide to said ribonucleotide to provide thestructure

d) selectively removing PG₃ from the 5′-hydroxyl of said2′-deoxyribonucleotide in pre-selected areas to provide a plurality offree 5′-hydroxyl groups; e) reacting said free 5′-hydroxyl groups withan additional 2′-deoxyribonucleotide having the structure

to yield a product of the structure

f) repeating steps d and e one or more times to provide saidoligonucleotides attached to said solid substrate; g) removing PG₂ fromone or more of said ribonucleotides to provide a free 2′-hydroxyl groupon each of said one or more ribonucleotides; and h) transesterifyingeach of said one or more ribonucleotides to yield said solid substratehaving a cyclic ester attached thereto and free oligonucleotides, eacholigonucleotide having a 3′-hydroxyl group and having the structure


2. A method according to claim 1 wherein PG₂ is acetate.
 3. A methodaccording to claim 1 wherein PG₃ or PG₄ is a photolabile protectinggroup.
 4. A method according to claim 3 wherein said photolabileprotecting group is selected from the group consisting of NNPOC andMBPMOC wherein said photolabile protecting group is attached to the5′-hydroxyl group of said 2′-deoxyribonucleotide as depicted by


5. A method according to claim 1 wherein the 2′-deoxyribonucleotide hasa phosphoramidite reactive group as shown by

wherein R₁ is selected from cyanoethyl, methyl, t-butyl, trimethylsilylor the like, and R₂ and R₃ are independently selected from isopropyl,cyclohexyl or the like.
 6. A method according to any of claims 1-5wherein B of the 2′-deoxyribonucleotide is selected from the groupconsisting of G, A, T, and C.
 7. A method according to claim 1 whereinPG₃ or PG₄ is an acid labile protecting group.
 8. A method according toclaim 7 wherein PG₃ or PG₄ is dimethoxytrityl (DMT).
 9. A methodaccording to claim 8 wherein the DMT group is removed in selected areasby exposure to acid generated by a photoacid generator in the presenceof electro magnetic radiation of an appropriate wavelength in thepresence of an acid scavenger.
 10. A method according to claim 9 whereinsaid acid scavenger is selected from the group consisting of organicbases and polymeric bases.
 11. A method according to claim 10 whereinsaid acid scavenger is a polymeric base.
 12. A method according to claim1 wherein said plurality of oligonucleotides comprises between 10¹ to10⁵ sets of different sequences, each distinct set of sequencescomprising a set.
 13. A method according to claim 1 wherein saidoligonucleotides are between about 80 to about 160 nucleotides.
 14. Amethod according to claim 1 wherein said density is between 200-2000pmol/cm².
 15. A method according to claim 1 wherein saidtransesterification is accomplished by raising the pH of the solution tobetween 9 and
 12. 16. A method according to claim 1 wherein saidtranesterification is initiated by the addition of metal ions.
 17. Amethod according to claim 1 wherein PG₂ is selected from the groupconsisting of FPMP, CEE, TBDMS and TOM.
 18. A method according to claim1 wherein said oligonucleotides are probes.
 19. A method according toclaim 1 wherein said oligonucleotides are primers.
 20. A methodaccording to claim 1 wherein between steps d and e unreacted5′-hydroxyls are capped.
 21. A method according to claim 1 wherein saiddesired array has between 10¹ to 10⁵ oligonucleotides of differentsequences and between about 80-160 bases in length.
 22. A methodaccording to claim 1 wherein said release oligonucleotides haveauthentic 3′-hydroxy termini upon exposure to a distinct set ofconditions or are further processed to have free 3′ hydroxyl termini.23. A method according to claim 1, further comprising the use of thephotoprotective groups NPPOC or NNPOC to provide suitable primer purityand quantity.
 24. A method according to claim 1 wherein DMT-basedphotoresist groups are used to provide fidelity primers.
 25. A methodaccording to claim 1 wherein ink-jet based in situ oligonucleotidesynthesis is used to provide an oligonucleotide probe.
 26. A methodaccording to claim 1 wherein synthesis is initiated with areverse-orientation RNA monomer that contains an orthogonal 2′-OHprotecting group.
 27. A method according to claim 7 wherein followingconventional 3′→5′ probe synthesis, the 2′-OH protecting group isremoved to allow base-induced intramolecular transesterification.
 28. Amethod according to claim 8 wherein the rate of transesterification isenchanced by raising the pH of the aqueous solution to pH 9-12 or by theaddition of particular metal ions.